Medical diagnostic assays often measure levels of antibodies against various proteins. Arrays of peptides may be used to simultaneously assay many different antibodies. But most antibodies bind to sequence-discontiguous structural epitopes.
Measurements of antibody (Ab) levels in blood and other tissues are widely used for screening and diagnosis of infectious diseases and many other medical conditions. Such tests may be used for public health screening or as part of a biodefense response architecture. The immune system produces antibodies (Abs) each with selective binding to particular proteins (or other biomolecules) called antigens. Abs are themselves proteins, and may be specified by their sequence. Each such specific Ab that is part of an effective immune response to a protein target antigen recognizes a particular epitope, a part of the target protein structure, typically on the exposed surface of the protein. The target proteins are themselves linear polypeptides, but they are folded into complex structures and may form complexes with other polypeptide chains. Epitopes may consist of extended linear stretches of the target protein sequence, but more often are composed of spatially adjacent discontiguous subsequences of the target protein or target protein complex, each subsequence a few amino acids in length (e.g., five amino acid residues). These spatially adjacent discontiguous subsequences are referred to as structural epitopes, as opposed to the less common extended linear epitopes.
In order to quickly and cheaply assay a single sample (e.g., assaying a blood sample for Abs against multiple different pathogens, as one would want to when screening people for exposure after a putative biological weapons attack) various formats may be used to simultaneously screen for binding of Abs to many different antigens. One format is an array of immobilized protein antigens, each antigen in a small spot in unique location in the array, so that reading out the location of antibody binding events allows the antibody binding specificity profile to be assessed (e.g., thus, in the example above, the target organism). Such protein arrays are costly, have limited chemical and thermal stability. Also, proteins may not be sufficiently densely immobilized or present the correct epitope structure, decreasing their sensitivity.
An alternative to protein arrays is to use arrays of short linear peptides. Compared to protein antigens, these are more chemically stable and less expensive, if they are relatively short (i.e., less than 20 amino acid residues). Such peptides may be immobilized or synthesized in situ at high density with extremely high sequence diversity: arrays may have >1 million different sequences. Various experimental and computational strategies may be used to select the peptide sequences, including purely random sequences, followed by purely empirical characterization of Ab binding profiles of various serotypes. However, it is desirable to find a design procedure that exploits the extensive genomics-based knowledge of actual or putative target protein sequences. (The combinatorial diversity of peptides is such that no chip however large will ever by able to comprehensively sample it). For example, one procedure is to simply take all short (e.g., 15 amino acid long) subsequences from each antigen protein and synthesize the corresponding peptides for use on chip.
One problem with using short linear subsequences of peptide arrays is that most epitopes are not linear subsequences, and the physical size of the binding site on the Ab molecule is such that it generally cannot contact more than a few amino acids of a peptide in extended conformation. Because Abs that recognize protein antigens generally recognize protein structures composed of co-localized but discontiguous patches of amino acid sequence (see above) linear peptides “tiled” from the protein sequence generally will not fully occupy the Ab binding site, leading to weaker binding of the Ab to the peptide compared to the protein epitope.